Synthesis and cytotoxic activity of pyridazino[1′,6′:1,2]pyrido[3,4-b]indol-5-inium derivatives as anti-cancer agents

Article abstract of DOI:10.1016/S0960-894X(02)00472-9

Several new pyridazino[1′,6′:1,2]pyrido[3,4-b]indol-5-inium derivatives were synthesised from β-carboline derivatives and their cytotoxic activity and effect on the cell cycle were evaluated against L1210 cancer cells.

Full text of DOI:10.1016/S0960-894X(02)00472-9

Bioorganic & Medicinal Chemistry Letters 12 (2002) 2611–2614
Synthesis and Cytotoxic Activity of Pyridazino[10,60:1,2]
pyrido[3,4-b]indol-5-inium Derivatives as Anti-Cancer Agents
Alberto Fontana,a Enrique J. Benito,a M. Justina Martın,a Nuria Sanchez,a
Ramon Alajarın,a J. Jose Vaquero,a Julio Alvarez-Builla,a,* Stephanie Lambel-Giraudet,b
Stephane Leonce,b Alain Pierreb and Daniel Caignardb
a
´
´
´
´
Departamento de Quımica Organica, Universidad de Alcala, 28871-Alcala de Henares, Madrid, Spain
bInstitut de Recherche Servier, 11 rue des Molineaux, 92450 Suresnes, France
Received 26 February 2002; revised 17 May 2002; accepted 17 June 2002
Abstract—Several new pyridazino[10,60:1,2]pyrido[3,4-b]indol-5-inium derivatives were synthesised from b-carboline derivatives and
their cytotoxic activity and effect on the cell cycle were evaluated against L1210 cancer cells. # 2002 Elsevier Science Ltd. All rights
reserved.
The term intercalator is applied to those compounds hav-
ing a planar chromophore capable of stacking between the
DNA base pairs.1,2 Intercalation provokes conformational
changes in the double helix, with consequences on the
normal mechanisms of DNA replication, transcription,
and repair.3 Interferences with these functions usually
results in non-specific cell killing.4 Thus, potential appli-
cations can be devised in the field of antitumor drugs.5ꢀ7
pyridazino[10,60:1,2]pyrido[3,4-b]indol-5-inium deriva-
tives and their in vitro antiproliferative activity against
L1210 leukaemia cell line. Starting from commercially
available b-carboline derivatives harmane (1), harmol (2)
or harmine (3), a series of compounds 4 (harmane series), 5
(harmol series) and 6 (harmine series) were prepared, with
variations at the domains 1 (bromo, nitro, amino, alkoxy
or hydroxy), 2 (carbamate, ester, amide, aliphatic satu-
rated or unsaturated) and 3 (aliphatic saturated, unsatu-
rated or aromatic) (Fig. 2).
A class of DNA intercalating agents is constituted by
charged molecules, with azinium8 and quinolizinium9ꢀ13
salts being the most representative examples of this kind
of polycyclic cations (Fig. 1). Our interest in the field
focussed our studies on DNA intercalators having a
bridgehead quaternary nitrogen, and led us to prepare
some benzimidazolium9,10 and g-carbolinium11ꢀ13
cations, which showed intercalating properties10ꢀ13 and
in vitro antiproliferative activity.9
The very related pyridazino[10,60:1,2]pyrido[3,4-b]indol-
5-inium system has been also previously prepared in our
laboratory by condensation of a b-carbolinium salt with
1,2-dicarbonyl14 derivatives (Westphal reaction). The
method was applied to the synthesis of the naturally
occurring alkaloid flavocorylene and related zwitter-
ionic indolo[2,3-a]quinolizinium compounds.15
Figure 1. General structure for azinium and quinolizinium DNA
intercalating agents.
Continuing our work in the field, we describe in
this communication the synthesis of several new
Figure 2. Starting materials 1–3 and general structure and domains
explored for compounds 4–6.
*Corresponding author. Tel.: +34-91-885-4606; fax: +34-91-885-
4686; e-mail: julio.alvarez@uah.es
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00472-9

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A. Fontana et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2611–2614
From 1, bromo or nitro derivatives 7 were pre-
pared.16,17 On the other hand, 1 was alkylated at the
indolic nitrogen to give 8. Amination of 7 and 8 with
O-(mesitylsulfonyl)hydroxylamine (MSH)18,19 gave
N-amino-b-carbolinium derivatives 9, which then were
reacted with 1,2-diketones through a Westphal reac-
tion, to give 4a–c and 4f–h. Some 4 derivatives, unsub-
stituted at the indolic position, were then treated with
base to give the ylide 10, which was then alkylated to give
4d–e. Finally, for 4h, reduction with stannous chloride
yielded 4i (Scheme 1 and Table 1).
with an aliphatic chain bearing an ester group (6i–k)
then, alkylation must be carried out after Westphal
reaction, otherwise this reaction would completely fail.
(Scheme 3 and Table 1).
From 2, indolic nitrogen was first protected as terc-
butoxy carbamate 11. Amination with MSH yielded 12,
which was then reacted with 3,4-hexanedione under
Westphal process to give 13. Carbamate was depro-
tected in acidic media to yield 5a. On the other hand, 11
was reacted through a Williamson reaction with several
alkyl halides to give 14. Further reaction with MSH to
give 15 and then, Westphal reaction with several
1,2-diketones led to 5b–l. For compounds 5k and 5l,
treatment with hydrobromic acid yielded 5m and 5n
(Scheme 2 and Table 1).
The last series were prepared from 3. First, alkylation at
the indolic nitrogen gave 16, which was then reacted
with MSH to give 17. On the other hand, 3 was ami-
nated by MSH to yield 18.15 Then, Westphal reaction
was performed on 17 and 18 to give 6c–g and 6a,b
respectively. Finally, compounds 6a and 6b were treated
with base to give 19 which was then alkylated to yield
6h–k. When the indolic nitrogen in 6 was substituted
Scheme 2. Reagents and conditions: (a) Boc2O, Et3N, DMAP,
CH2Cl2; (b) MSH, CH2Cl2; (c) 1,2-diketone, Et3N, EtOH, reflux; (d)
HBr, acetone, reflux; (e) RX, KOH, K2CO3, MeCN.
Scheme 1. Reagents and conditions: (a) Br2, THF or HNO3; (b) RX,
KOH, K2CO3, MeCN; (c) MSH, CH2Cl2; (d) 1,2-diketone, Et3N,
EtOH, reflux; (e) Et3N, H2O; (f) R1X, mw, 10 min, 300 watt, two runs
or R1X, CH2Cl2; (g) SnCl2, HCl, reflux.
Scheme 3. Reagents and conditions: (a) RX, KOH, K2CO3, MeCN;
(b) MSH, CH2Cl2; (c) 1,2-diketone, Et3N, EtOH, reflux; (d) Et3N,
H2O; (e) R1X, mw, 10 min, 300 watt, two runs or RX, CH2Cl2.

A. Fontana et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2611–2614
Table 1. Cytotoxicity and cell cycle effects against L1210 leukaemia for compound 1
2613
Compd
R
R1
R2
IC50 (mM)
Cell cyclea
Concn (mM)
b
G1
G2Mc
4a
4b
4c
4d
4e
4f
4g
4h
4i
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
6a
6b
6c
6d
6e
6f
6g
H
H
H
H
H
9-Br
(CH2)3CCH
(CH2)3CCH
CH2CO2Et
CH2CONHCH2CCH
1,8-Dinaphthyl
Et
1,8-Dinaphthyl
1,8-Dinaphthyl
>4
6.4
1.2
10.4
1.9
2.3
15.3
1.5
1.0
10.0
6.7
0.5
+
+
1d
25d
10d
50d
20d
10d
50
5f
NSe
NS
NS
NS
NS
NS
NS
NS
56
NS
NS
NS
NS
NS
NS
NS
NS
(CH2)3Br
H
Et
Et
Et
Et
Et
Et
Me
Et
Et
Me
Et
Me
Et
Et
Et
Me
9-NO2
H
H
H
H
Boc
Boc
Boc
Boc
Boc
Boc
Boc
Boc
Boc
Boc
Boc
H
H
H
H
Me
11-NO2
11-NH2
10-OH
5f
10
50
5
5
10
5
5
2
2
0.5
10
2.5
10
20
5
10-OC2H5
10-O-C2H5
10-O-nC3H7
10-O-nC4H9
10-O-nC4H9
10-O-nC5H11
10-O-nC5H11
10-O-nC7H15
10-O-nC10H21
10-OBn
10-OBn
10-OBn
10-OBn
10-OMe
10-OMe
10-OMe
10-OMe
10-OMe
10-OMe
10-OMe
10-OMe
10-OMe
10-OMe
10-OMe
64
0.46
1.3
NS
NS
+
NS
+
62
63
NS
59
NS
NS
0.36
0.23
0.28
0.13
0.17
2.3
0.9
5.2
1.9
1.3
2.0
2.3
0.048
0.065
1.3
72.9
1.1
5.0
1.1
NS
NS
NS
Et
Me
Et
Me
NS
+
38
35
Et
(i-Pr)3SiCCH
5
NEg
57
60
+
NE
+
NS
nC5H11
nC5H11
(CH2)3CCH
CH2CONH CH2CCH
Me
Me
Et
2-Furyl
Et
Et
Me
Et
Et
1
1
5d
6h
6i
6j
6k
10d
50
5
10
0.1
0.05
CH2CO2Et
CH2CO2Et
(CH2)3CO2Et
NS
++
NS
81
2.0
0.025
0.03
NS
Adriamycin
Camptothecin
80
aPercent of untreated control L1210 cells in the phases of the cell cycle: 41% (G1), 24% (G2M).
bPercent of treated cells in the G1 phase: (+) 54–64%; (++) 65–75%.
cPercent of treated cells in the G2M phase: (+) 35–45%; (++) 46–66%.
dToxic.
eNS, not significant.
f30% apoptosis (10 mM).
gNE, not evaluated.
As a result, a series of new pyridazino[10,60:1,2]pyrido[3,4-
b]indol-5-inium derivatives have been prepared and their
cytotoxic activity tested in vitro against L1210 leukaemia
and for their effect on the L1210 cell cycle. Two com-
pounds, 6d and 6e showed activity at the same level than
adriamycin and camptothecin, and exert their effect in the
G1 phase of the cell cycle.
Cytotoxic activity
Compounds 4–6 were tested in vitro against L1210 leu-
kaemia and for their effect on the L1210 cell cycle.20,21
Results are reported in Table 1.
The activity data show that compounds 5i and 5j were
the most active in the harmol series. In the harmine
series compounds 6d and 6e were the most active pre-
pared so far. They showed good activity as compared to
adriamycin and camptothecin.22 From cytotoxicity data
it can be deduced than a lypophillic side chain, more
than seven atoms long at C10-position is preferred in
domain 1 (5i, 5j). For domain 2, lypophillic chains at
least five atoms long give better activities (6d, 6e). In
domain 3, aliphatic groups are preferred. On the other
hand, it is interesting to notice than the most cytotoxic
compounds have effect in the G1 phase of the cell cycle
while the reference compounds exert their effect in the
G2M phase.
Acknowledgements
The authors wish to acknowledge the Comision Inter-
ministerial de Ciencia y Tecnologıa (CICYT, Project
SAF98–0093) and SERVIER for financial support.
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22. 6d: 1H NMR (300 MHz, DCCl3) d 8.98 (d, 1H, J=7.0
Hz); 8.65 (s, 1H); 8.56 (d, 1H, J=7.0 Hz); 8.19 (d, 1H, J=8.7
Hz); 7.23 (s, 1H); 7.04 (d, 1H, J=8.7 Hz); 6.71 (s, 2H);
4.85–4.62 (m, 2H); 3.97 (s, 3H); 2.74 (s, 3H); 2.67 (s, 3H); 2.49
(s, 6H); 2.11 (s, 3H); 1.89–1.86 (m, 2H); 1.42–1.25 (m, 4H);
0.84 (t, 3H, J=6.8 Hz). Anal. calcd for C31H37N3O4S, C:
61.88; H: 7.20; N: 6.98; found C: 61.74; H: 7.37; N: 7.07. 6e:
1H NMR (300 MHz, DCCl3) d 9.13 (d, 1H, J=7.2 Hz);
8.67–8.65 (m, 2H); 8.31 (d, 1H, J=8.7 Hz); 7.37 (s, 1H); 7.16
(d, 1H, J=8.7 Hz); 6.78 (s, 2H); 4.96–4.93 (m, 2H); 4.04 (s,
3H); 3.23 (c, 2H, J=7.0 Hz); 3.07 (c, 2H, 7.2 Hz); 2.55 (s, 6H);
2.18 (s, 3H); 2.05–1.99 (m, 2H); 1.57–1.48 (m, 10H); 0.93 (t,
3H, J=7.2 Hz). Anal. calcd for C33H41N3O4S. 2H2O, C:
66.75; H: 7.30; N: 7.08; found C: 66.97; H: 7.31; N: 6.85.
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